Tóth K, Vaughan M M, Slocum H K, Arredondo M A, Takita H, Baker R M, Rustum Y M
Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, NY 14263.
Am J Pathol. 1994 Feb;144(2):227-36.
We have developed a new immunoperoxidase "sandwich" staining method for amplified detection of P-glycoprotein (Pgp) that is suitable for use on formalin-fixed, paraffin-embedded (conventional) tissue sections. This was accomplished by substantially changing the procedure described by Chan (1988) so as to increase specific staining intensity and to decrease nonspecific background staining. To determine the most appropriate primary antibody for the assay, we compared the immunoreactivity of JSB-1, C494, and C219 monoclonal antibodies recognizing internal epitopes of Pgp, and MRK16 and 4E3 monoclonal antibodies recognizing external epitopes of Pgp. Paraffin sections of Pgp-positive normal human tissues (adrenal, liver, kidney, and brain), of renal tumors, and of cell pellets of sensitive and multidrug resistant human tumor cell lines (MCF-7, KB) were used for comparisons. Immunostaining was excellent with JSB-1, moderate with C494, and very weak with C219. MRK16 and 4E3 showed no reaction. Nonspecific background staining was reduced by 1) omitting immunoglobulin G from secondary antibodies; 2) decreasing the concentration of peroxidase-antiperoxidase complex; and 3) utilizing casein solution for blocking and washing. Pretreatment of sections before immunostaining was also simplified. Using JSB-1, the threshold for detection of elevated Pgp corresponded to less than two-fold relative resistance to doxorubicin. Applying this method, we found two of 26 non-small cell lung cancers were positive for Pgp, consistent with previous results of others using frozen sections. This new immunoperoxidase sandwich staining method using JSB-1 now allows reliable Pgp detection in sections of formalin-fixed, paraffin-embedded (archived) surgical specimens and small biopsy materials commonly used for diagnostic purposes.
我们开发了一种新的免疫过氧化物酶“夹心”染色方法，用于扩增检测P-糖蛋白（Pgp），该方法适用于福尔马林固定、石蜡包埋（传统）的组织切片。这是通过大幅改变Chan（1988年）描述的程序来实现的，以提高特异性染色强度并减少非特异性背景染色。为了确定该检测最合适的一抗，我们比较了识别Pgp内部表位的JSB-1、C494和C219单克隆抗体，以及识别Pgp外部表位的MRK16和4E3单克隆抗体的免疫反应性。使用Pgp阳性的正常人组织（肾上腺、肝脏、肾脏和脑）、肾肿瘤以及敏感和多药耐药的人肿瘤细胞系（MCF-7、KB）的细胞沉淀的石蜡切片进行比较。JSB-1的免疫染色效果极佳，C494的效果中等，C219的效果非常弱。MRK16和4E3无反应。通过以下方法减少了非特异性背景染色：1）在二抗中省略免疫球蛋白G；2）降低过氧化物酶-抗过氧化物酶复合物的浓度；3）使用酪蛋白溶液进行封闭和洗涤。免疫染色前切片的预处理也得到了简化。使用JSB-1时，检测Pgp升高的阈值对应于对阿霉素的相对耐药性低于两倍。应用该方法，我们发现26例非小细胞肺癌中有两例Pgp呈阳性，这与其他人使用冰冻切片的先前结果一致。这种使用JSB-1的新免疫过氧化物酶夹心染色方法现在能够在福尔马林固定、石蜡包埋（存档）的手术标本切片和通常用于诊断目的的小活检材料中可靠地检测Pgp。
